Triple-negative breast cancers (TNBC) are highly aggressive with poor prognosis and resistance to available therapies. TRAIL (TNF-related apoptosis-inducing ligand) is a promising cancer therapeutic agent due to its minimal toxicity to normal tissues and remarkable apoptotic activity in tumors. However, most breast cancer (BC) cells are resistant to TRAIL-induced apoptosis. Using a genomewide siRNA screening approach, we unexpectedly identified androgen receptor (AR) to be responsible for TRAIL resistance. Our immediate objective is to define the function and mechanisms of AR in the development of TRAIL resistance in BC. Our long-term goal is to determine whether AR provides an effective target for making TNBC cells sensitive to TRAIL-induced apoptosis, providing a new therapeutic strategy for this deadly subset of BC. In our preliminary studies, we found that AR expression levels were markedly elevated in human invasive BC including TNBC specimens. Importantly, BC cell lines express different levels of AR that correlated with their TRAIL resistance. AR overexpression in TNBC cells protected against TRAIL-induced apoptosis whereas knockdown or inhibition of AR sensitized BC cells to TRAIL. Further, we observed an upregulation of the TRAIL receptor, Death Receptor 5 (DR5), following the removal or inhibition of AR. We thus hypothesize that AR signaling suppresses TRAIL-induced BC cell apoptosis, in part, by suppressing DR5 expression, and that a combination of AR inhibitors with TRAIL may be a novel and effective therapy for TNBC. We will test this hypothesis using molecular, cellular and animal models. Aim 1: To assess the pathological importance of AR in TNBC. We will silence AR in TRAIL- resistant TNBC cells (MDA-MB-468 and BT20) or overexpress AR in TRAIL-sensitive TNBC cells (MDA-MB-231 and -436). The effects of altering AR expression on growth and TRAIL-induced apoptosis of these stable cell lines will be examined in culture and in an orthotopic xenograft mouse model. Aim 2: To determine the molecular mechanisms of AR suppression of TRAIL signaling. TRAIL induces the formation of the Death Inducing Signaling Complex (DISC) including DR5, FADD, and caspase- 8. We will first determine the effects of altering AR expression on DR5 expression in BC cells. We will then determine if over-expression of DR5 is sufficient to overcome AR-induced TRAIL resistance. We will also use mutagenesis to identify domains of AR critical for regulation of DR5 and TRAIL resistance. Whether AR interferes with the TRAIL pathway by directly interacting with the components of the DISC will be examined. Further, we will investigate the role of the mitochondrial apoptotic pathway in AR-loss induced sensitization of BC cells to TRAIL. Aim 3: Targeting AR to improve the effectiveness of TRAIL-therapy against TNBC. Since AR antagonists potentiate TRAIL-induced apoptosis in TNBC cells, we will examine the effects of AR antagonists in combination with TRAIL on TNBC growth in an orthotopic xenograft mouse model. These studies will determine whether AR provides an effective target for treatment of TRAIL-resistant TNBC.